CN107505427B - Thin-layer chromatography detection method for traditional Chinese medicine rheum palmatum - Google Patents
Thin-layer chromatography detection method for traditional Chinese medicine rheum palmatum Download PDFInfo
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- 239000000523 sample Substances 0.000 claims description 9
- 238000011161 development Methods 0.000 claims description 8
- 229910021529 ammonia Inorganic materials 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 238000002481 ethanol extraction Methods 0.000 claims description 4
- PQPKPCPUSAPERC-UHFFFAOYSA-N ethyl acetate;formic acid;hexane Chemical compound OC=O.CCCCCC.CCOC(C)=O PQPKPCPUSAPERC-UHFFFAOYSA-N 0.000 claims description 4
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 3
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- WBJINCZRORDGAQ-UHFFFAOYSA-N formic acid ethyl ester Natural products CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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Abstract
The invention discloses a detection method of traditional Chinese medicine rheum palmatum, which adopts thin-layer chromatography for detection and comprises the following steps: (1) preparing a test solution: extracting the test medicinal material powder with ethanol, filtering, concentrating, dissolving in water, adding organic solvent for extraction, concentrating, and diluting with methanol to desired volume to obtain test solution; (2) and (3) thin-layer chromatography detection: spotting the sample solution on a thin layer plate, developing with petroleum ether 30-60 deg.C-n-hexane-ethyl acetate-formic acid as developing agent, developing, inspecting, and comparing. According to the thin-layer chromatography detection method, purple characteristic spots exist in the Rf value range of 0.13-0.17, the spots are unique to rheum palmatum, are clear and easy to distinguish, and can be used for effectively identifying rheum palmatum from rheum palmatum, rheum tanguticum and rheum officinarum.
Description
Technical Field
The invention relates to a thin-layer chromatography detection method for identifying characteristics of traditional Chinese medicine rheum palmatum.
Background
The thin layer chromatography is a method for identifying, checking impurities or measuring the content of a drug by spotting a sample solution on a thin layer plate, developing the sample solution in a developing container by using a developing agent, and comparing the obtained chromatogram with a chromatogram obtained by using a suitable standard substance according to the same method. The different adsorption capacity of each component to the same adsorbent is utilized, so that adsorption, desorption, re-adsorption and re-desorption are continuously generated in the process that the solvent flows through the adsorbent, and the components contained in the sample are separated. Thin-layer chromatography has the advantages of simple equipment, simple and convenient operation, high separation speed, high sensitivity and resolution and the like, is used for the aspects of impurity detection, drug analysis and the like in medicines by a plurality of national formulary, is one of the most accepted methods for identification and related substance detection in the current formulary, and is an important experimental technique for quickly separating and qualitatively analyzing a small amount of substances (Yi Li, Zong lan, Puxiao hui, and the like.
Rhubarb is a traditional Chinese medicine in China, which is recorded as the book of Shen nong herbal medicine, has the efficacies of purging heat, relaxing bowels, cooling blood, removing toxicity, dispelling blood stasis, dredging channels and the like, is commonly used for treating excess heat constipation, abdominal pain due to stagnation, diarrhea and dysentery discomfort, damp-heat jaundice and the like in clinic, and is widely applied. With the increase of clinical medication and limited medicinal material resources, commercial rhubarb in the market has uneven quality, and even counterfeit rhubarb is regarded as genuine rhubarb for medication. There are three sources of genuine rhubarb collected in the first department of the pharmacopoeia of China 2015 edition, which are the dried roots and rhizomes of Rheum palmatum L.var.palmatum L.var.maxim, Rheum tanguticum Maxim or Rheum officinale Baill. However, in order to better control the quality of the medicine in the actual process of preparing the traditional Chinese medicine, the source of crude medicine decoction pieces needs to be controlled, and if the raw material medicine of a certain preparation is only rhubarb, the characteristic identification of the rhubarb is required to avoid being confused with other two basic sources.
At present, no effective method for identifying the rhubarb palmate, the rhubarb tangkuei and the rhubarb horsetails is reported at home and abroad.
Disclosure of Invention
In order to solve the problems, the invention provides a thin-layer chromatography detection method of a traditional Chinese medicine rheum palmatum.
The invention provides a detection method of traditional Chinese medicine rheum palmatum, which adopts thin-layer chromatography for detection and comprises the following steps:
(1) preparing a test solution: extracting the test medicinal material powder with ethanol, filtering, concentrating, dissolving in water, adding organic solvent for extraction, concentrating, and diluting with methanol to desired volume to obtain test solution;
(2) and (3) thin-layer chromatography detection: spotting the sample solution on a thin layer plate, developing with petroleum ether 30-60 deg.C-n-hexane-ethyl acetate-formic acid as developing agent, developing, inspecting, and comparing.
Wherein in the step (1), the volume-to-mass ratio of the ethanol to the test medicine powder is 40:3 ml/g; and/or the volume mass ratio of the water to the test medicine powder is 40:3 ml/g; and/or the volume mass ratio of the organic solvent to the test medicine powder is 20:1 ml/g; and/or the volume mass ratio of the methanol to the test medicine powder is 4:3 ml/g.
Wherein in the step (1), the ethanol extraction is carried out for 2 times and each time for 15 min; and/or the organic solvent extraction is that the organic solvent is shaken and extracted for 2 to 5 times; and/or the ethanol is 85% ethanol, 95% ethanol or absolute ethanol; and/or the ethanol extraction is ultrasonic extraction.
Preferably, the number of times of shaking extraction of the organic solvent is 2.
Preferably, the ethanol is 85% ethanol.
Wherein in the step (1), the organic solvent is dichloromethane, trichloromethane or ethyl acetate; preferably, the organic solvent is dichloromethane.
Wherein, in the step (2), the thin-layer plate is a silica gel H plate which takes sodium carboxymethyl cellulose as an adhesive, and/or in the step (2), the sample application amount of the sample application is 2-5 mul.
In the step (2), the volume ratio of the developing agent petroleum ether to the n-hexane-ethyl acetate-formic acid is 3-7: 11-12: 5-8: 0.2; preferably, the volume ratio of the developing solvent petroleum ether to the n-hexane-ethyl acetate-formic acid is 4-5: 12: 6-8: 0.2; more preferably, the volume ratio of the developing agent petroleum ether 30-60-n-hexane-ethyl acetate-formic acid is 4:12:6: 0.2.
In the step (2), the unfolding distance of the unfolding is 7-9 cm; preferably, the spread distance of the spread is 8 cm.
Wherein in the step (2), the color development is an ammonia vapor color development method; the thin layer chromatography detection shows that purple characteristic spots exist in the Rf value range of 0.13-0.17.
The invention has the beneficial effects that: the invention provides a thin-layer chromatography detection method for traditional Chinese medicine rheum palmatum, which has good development effect, has unique characteristic spots of rheum palmatum, is clear and easy to distinguish, and can effectively identify the rheum palmatum from rheum palmatum, rheum tanguticum and rheum officinarum.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a thin-layer chromatogram for identifying Rheum palmatum, Rheum tanguticum Maxim and Rheum officinale Kitag of the present invention, wherein 1-2 are Rheum palmatum comparison medicinal materials, 3-4 are Rheum tanguticum Maxim comparison medicinal materials, 5-6 are Rheum officinale Kitag comparison medicinal materials, and 7-8 are blank solvents;
FIG. 2 is a thin layer chromatogram with an developed system of acetone-methanol-n-hexane-dichloromethane (1:1:1: 1);
FIG. 3 is a thin layer chromatogram with an evolving system of ethyl acetate-absolute ethanol-n-hexane-dichloromethane (1:1:1: 1);
FIG. 4 is a thin layer chromatogram with an expanded system of n-hexane-ethyl acetate-methanol-glacial acetic acid (20:25:5: 2);
FIG. 5 is a thin layer chromatogram with petroleum ether (30-60 ℃ C.) -n-hexane-ethyl acetate-formic acid (4:12:6:0.2) as an evolving system; .
FIG. 6 is a thin layer chromatogram with an expanded system of n-hexane-ethyl acetate-glacial acetic acid (9:2: 0.6);
FIG. 7 is a thin-layer chromatogram of the sample amount under investigation, wherein 1-2 is radix et rhizoma Rhei Palmati reference medicinal material 1 μ l, 3-4 is radix et rhizoma Rhei Palmati reference medicinal material 2 μ l, 5-6 is radix et rhizoma Rhei Palmati reference medicinal material 3 μ l, and 7-8 is radix et rhizoma Rhei Palmati reference medicinal material 5 μ l;
FIG. 8 is a thin layer chromatogram with an expansion distance of 7 cm;
FIG. 9 is a thin layer chromatogram with an expansion distance of 8 cm;
FIG. 10 is a thin layer chromatogram with an expansion distance of 9 cm.
Detailed Description
1. Instrument for measuring the position of a moving object
Thin-layer glass plate: 100 x 200 mm;
an ultrasonic instrument: a Tianjin Ottosain Instrument;
a water bath kettle: shanghai sperm macro experimental facility;
an electronic balance: shanghai sperm science and beauty instrument;
ultraviolet instrument: shanghai city Anting electronic instrument;
long-neck funnel, separating funnel, evaporating dish, and 200X 100mm chromatographic cylinder (vertical).
2. Reagent
Silica gel H board: qingdao maritime works 20160721;
acetone, methanol, n-hexane, dichloromethane, ethyl acetate, absolute ethanol, cyclohexane, petroleum ether (30-60 ℃), n-hexane, ethyl formate, formic acid, glacial acetic acid: all the materials are analytically pure and purchased from Chengdu Kelong chemical preparation factories.
3. Reagent
Radix et rhizoma Rhei Palmati reference (121249-201304); tanggute radix Et rhizoma Rhei reference drug (120902-; a medicinal rhubarb reference drug (120984-; the reference materials were purchased from the institute for food and drug testing, China.
Example 1 detection method of the invention
(1) Preparation of medicinal solution
Taking 0.75g of rhubarb powder to be detected, precisely weighing, placing in a conical flask with a stopper, adding 10ml of 85% ethanol, carrying out ultrasonic treatment for 2 times, 15 minutes each time, filtering, combining filtrates, evaporating the filtrates to dryness, adding 10ml of water into residues for dissolving, shaking and extracting for 2 times, 15ml each time, combining dichloromethane solutions, evaporating to dryness, adding 1ml of methanol into the residues for dissolving, and taking the residues as a sample solution.
(2) Thin layer chromatography detection
Sucking 3 μ l of the sample solution, dropping on silica gel plate with methanol as blank solvent, developing with petroleum ether (30-60 deg.C) -n-hexane-ethyl acetate-formic acid (4:12:6:0.2) as developing agent, taking out, air drying, fumigating in ammonia vapor, and inspecting under sunlight.
(3) Result judgment
If there is purple characteristic spot in Rf value of 0.13-0.17, it is judged as Rheum palmatum, and if there is no purple characteristic spot in Rf value of 0.13-0.17, it is judged as other Rheum officinale.
The following experimental examples are provided to illustrate the advantageous effects of the present invention.
Experimental example 1 identification of Rheum palmatum L
(1) Preparation of medicinal solution
Respectively taking 0.75g of powder of rheum palmatum, rheum tanguticum and medicinal rhubarb, precisely weighing, placing in a conical flask with a stopper, adding 10ml of 85% ethanol, carrying out ultrasonic treatment for 2 times, 15 minutes each time, filtering, combining filtrates, drying the filtrate by distillation, dissolving the residue by adding 10ml of water, extracting by shaking dichloromethane for 2 times, 15ml each time, combining dichloromethane solutions, drying by distillation, and dissolving the residue by adding 1ml of methanol to obtain a sample solution.
(2) Thin layer chromatography detection
Sucking 3 mu l of each sample solution, taking methanol as a blank solvent, dropping the sample solution on a silica gel plate, developing the sample solution by using petroleum ether (30-60 ℃) and n-hexane-ethyl acetate-formic acid (4:12:6:0.2) as a developing agent, taking out the sample solution, drying the sample solution in the air, smoking the sample solution in ammonia steam, and viewing the product under sunlight, wherein a thin-layer chromatogram is shown in a figure 1, wherein the serial numbers 1-2 in the figure are rheum palmatum, 3-4 are rheum tanguticum, 5-6 are medicinal rheum officinale, and 7-8 are methanol.
(3) Results of the experiment
The results show that: the chromatogram map Rf value of the Rheum palmatum medicinal material is 0.13-0.17, and the Rheum tanguticum and the medicinal Rheum officinale medicinal material do not show purple characteristic spots at the corresponding positions of the Rheum palmatum medicinal material.
It can be seen that the target spot is sharp, well separated from other spots, and has no smearing.
Experimental results prove that the method can effectively distinguish the rheum palmatum from the rest rheum palmatum and can be used for identifying the rheum palmatum.
Experimental example 2 selection of developing agent for detection method of the present invention
(1) Preparation of reference drug solution
0.75g of powder of Rheum palmatum, Rheum tanguticum Maxim and Rheum officinale Baill were taken respectively, and control solutions were prepared according to the method of example 1.
(2) Thin layer chromatography detection
And (3) dropping 3 mu l of the solution on a silica gel H thin-layer plate using sodium carboxymethylcellulose as a binder, respectively developing by using a following development system, taking out, airing, fumigating in ammonia steam, and inspecting under sunlight, wherein the thin-layer chromatogram is shown in figures 2-6, wherein the serial numbers 1-2 in the figures are rheum palmatum, 3-4 in Tanggute rhubarb, and 5-6 in medicinal rhubarb.
And (3) unfolding the system I: acetone-methanol-n-hexane-dichloromethane (1:1:1: 1);
and (3) unfolding the system II: ethyl acetate-absolute ethyl alcohol-n-hexane-dichloromethane (1:1:1: 1);
and (3) unfolding the system III: n-hexane-ethyl acetate-methanol-glacial acetic acid (20:25:5: 2);
and (4) unfolding the system IV: petroleum ether (30-60 ℃) -n-hexane-ethyl acetate-formic acid (4:12:6: 0.2);
and (4) unfolding the system V: n-hexane-ethyl acetate-glacial acetic acid (9:2: 0.6).
(3) Results of the experiment
The results show that: the system I has poor spreading effect, each spot is converged at the front edge of the solvent, the trailing is serious, and the characteristic spots cannot be seen. System II characteristic spots were not separated from the spotting origin. The purple characteristic spots of Rheum palmatum were seen in both System III and System V, but with varying degrees of smearing. The purple characteristic spots of the rhubarb leaves of the system IV are clear and round, the separation degree from other spots is good, and no trailing phenomenon exists. System IV was therefore selected as the developing solvent for characterization of rheum palmatum.
The experimental results of the thin-layer chromatography detection of the development system I-V show that: only by selecting a specific developing agent petroleum ether (30-60 ℃) -n-hexane-ethyl acetate-formic acid for developing, the purpose that the rheum palmatum has purple characteristic spots, is clear and round, has good separation degree with other spots, has no trailing phenomenon and can be clearly distinguished from other rheum palmatum.
Experimental example 3 determination of sample application amount
(1) Preparation of medicinal solution
The powder of Rheum palmatum was used to prepare a control solution according to the method of example 1.
(2) Thin layer chromatography detection
Respectively sucking the above solutions 1 μ l, 2 μ l, 3 μ l and 5 μ l on the same silica gel plate, developing with petroleum ether (30-60 deg.C) -n-hexane-ethyl acetate-formic acid (4:12:6:0.2) as developing agent, taking out, air drying, fumigating in ammonia vapor, and inspecting in sunlight to obtain a thin layer chromatogram as shown in FIG. 7, wherein the numbers 1-2 in the figure are radix et rhizoma Rhei Palmati control solution 1 μ l, 3-4 are radix et rhizoma Rhei Palmati control solution 2 μ l, 5-6 are radix et rhizoma Rhei Palmati control solution 3 μ l, and 7-8 are radix et rhizoma Rhei Palmati control solution 5 μ l.
(3) Results of the experiment
The results show that: the spot size is in the range of 2-5 mul, the spot is clear, and no tailing exists.
Experimental example 4 determination of spreading distance
(1) Preparation of medicinal solution
The powder of Rheum palmatum was used to prepare a control solution according to the method of example 1.
(2) Thin layer chromatography detection
Dropping 3 μ l of the above solution on a silica gel plate, developing with petroleum ether (30-60 deg.C) -n-hexane-ethyl acetate-formic acid (4:12:6:0.2) as developing agent for 7cm, 8cm and 9cm respectively, taking out, air drying, fumigating in ammonia vapor, and inspecting under sunlight to obtain a thin layer chromatogram shown in fig. 8-10.
(3) Results of the experiment
The results show that: when the spreading distance is 8cm, the edge effect is small, the separation degree of each spot is good, and the spots are clear.
In conclusion, the sample extraction method provided by the invention utilizes ultrasonic extraction, reduces the sample preparation time, simplifies the operation steps, does not add acid hydrolysis in the extraction process, uses dichloromethane to replace chloroform for shaking extraction, and optimizes the extraction solvent; the thin-layer chromatography detection method for the rheum palmatum leaves has purple characteristic spots within the Rf value range of 0.13-0.17, has good development effect, is unique to the rheum palmatum leaves, is clear and easy to distinguish, and can effectively identify the rheum palmatum leaves from the rheum palmatum leaves, the rheum tanguticum and the rheum officinarum.
Claims (12)
1. A detection method of traditional Chinese medicine rheum palmatum is characterized by comprising the following steps: the detection is carried out by adopting a thin layer chromatography, and the steps are as follows:
(1) preparing a test solution: extracting the test medicinal material powder with ethanol, filtering, concentrating, dissolving in water, adding organic solvent for extraction, concentrating, and diluting with methanol to desired volume to obtain test solution; the organic solvent is dichloromethane, trichloromethane or ethyl acetate;
(2) and (3) thin-layer chromatography detection: spotting the sample solution on a silica gel H plate, developing with petroleum ether 30-60 deg.C-n-hexane-ethyl acetate-formic acid as developing agent, developing, inspecting, and comparing; the volume ratio of the developing agent petroleum ether to the n-hexane-ethyl acetate-formic acid is 3-7: 11-12: 5-8: 0.2.
2. The detection method according to claim 1, characterized in that: in the step (1), the volume-to-mass ratio of the ethanol to the test medicine powder is 40:3 ml/g; and/or the volume mass ratio of the water to the test medicine powder is 40:3 ml/g; and/or the volume mass ratio of the organic solvent to the test medicine powder is 20:1 ml/g; and/or the volume mass ratio of the methanol to the test medicine powder is 4:3 ml/g.
3. The detection method according to claim 1, characterized in that: in the step (1), the ethanol extraction is carried out for 2 times, and each time is 15 min; and/or the organic solvent extraction is that the organic solvent is shaken and extracted for 2 to 5 times; and/or the ethanol is 85% ethanol, 95% ethanol or absolute ethanol; and/or the ethanol extraction is ultrasonic extraction.
4. The detection method according to claim 3, characterized in that: the number of shaking extraction times of the organic solvent is 2; and/or the ethanol is 85% ethanol.
5. The detection method according to claim 1, characterized in that: in the step (1), the organic solvent is dichloromethane.
6. The detection method according to claim 1, characterized in that: in the step (2), the silica gel H plate is a silica gel H plate taking sodium carboxymethylcellulose as an adhesive; and/or in the step (2), the sample application amount of the sample application is 2-5 mu l.
7. The detection method according to claim 1, characterized in that: in the step (2), the volume ratio of the developing agent petroleum ether 30-60-n-hexane-ethyl acetate-formic acid is 4-5: 12: 6-8: 0.2.
8. The detection method according to claim 7, characterized in that: in the step (2), the volume ratio of the developing agent petroleum ether 30-60-n-hexane-ethyl acetate-formic acid is 4:12:6: 0.2.
9. The detection method according to claim 1, characterized in that: in the step (2), the unfolding distance of the unfolding is 7-9 cm.
10. The detection method according to claim 9, characterized in that: in the step (2), the unfolding distance of the unfolding is 8 cm.
11. The detection method according to claim 1, characterized in that: in the step (2), the color development is an ammonia vapor color development method.
12. The detection method according to claim 1, characterized in that: in the step (2), purple characteristic spots exist in the Rf value range of 0.13-0.17 by the thin layer chromatography detection.
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